Anti-pad2 antibody for treating and evaluating rheumatoid arthritis

ABSTRACT

A method for obtaining a prognosis for a subject having rheumatoid arthritis by measuring the levels of anti-PAD2 antibodies is described. Methods of using anti-PAD2 antibodies to treat rheumatoid arthritis are also described.

CONTINUING APPLICATION DATA

This application claims the benefit of U.S. Provisional Application Ser.No. 62/481,158, filed Apr. 4, 2017, the disclosure of which isincorporated by reference herein.

GOVERNMENT FUNDING

This invention was made with government support under grant number NIHNIAMS AR050026-10 awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

BACKGROUND

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterizedby immune-mediated damage of synovial joints that affects approximately1% of the population. There is marked heterogeneity in the clinicalpresentation, disease course, involvement of extra-articular organs, andresponse to therapy observed among individuals with RA, but themechanisms driving this diversity are poorly understood.Anti-citrullinated protein antibodies (ACPAs), detected by theanti-cyclic citrullinated peptide (CCP) assay, are hallmark serologicfeatures of patients with RA and serve as valuable diagnosticbiomarkers. Taylor et al., Autoimmune Dis; 2011:815038 (2011). ACPAs areassociated with specific HLA-DRβ1 alleles that confer genetic risk forRA development, collectively referred to as “shared epitope” (SE)alleles. Holoshitz J., Curr Opin Rheumatol, 22(3):293-298 (2010).Although ACPA-positive patients tend to have more severe disease onaverage than ACPA-negative individuals, the clinical heterogeneity inthis group precludes the use of ACPAs alone as reliable prognosticbiomarkers. Precise markers that specifically identify clinically uniquesubgroups may reveal distinct underlying disease mechanisms withdifferences in prognosis and response to treatment.

The citrullinated protein targets of ACPAs are generated through thecalcium-dependent deimination of arginine residues by thepeptidylarginine deiminase (PAD) enzymes. Witalison et al., Curr DrugTargets, 16(7):700-710 (2015). There are five members of the PAD enzymefamily (PAD1, PAD2, PAD3, PAD4, and PAD6), which display diverse tissuedistribution and substrate specificity. PAD2 and PAD4 are implicated ascentral drivers of RA pathogenesis. Polymorphisms in the padi2 and padi4genes are independently associated with RA development; PAD2 and PAD4are observed in the tissue and fluid of inflamed RA joints; and bothenzymes can generate citrullinated autoantigens. Darrah et al., AnnRheum Dis, 71(1):92-98 (2012). Interestingly, autoantibodies to PAD4 arepresent in ˜35% of patients with RA and are associated with ACPAs anderosive disease that persists despite treatment with TNFα inhibitors.Harris et al., Arthritis Rheum, 58(7):1958-1967 (2008). Moreover, asubgroup of anti-PAD4 antibodies that cross react with PADS (anti-PAD3/4antibodies) has recently been identified. These antibodies have thecapacity to activate the PAD4 enzyme by lowering the amount of calciumrequired for catalysis. Patients with anti-PAD3/4 antibodies have themost erosive RA that progresses despite treatment with standardtherapies and are at the highest risk of having RA-associatedinterstitial lung disease (ILD). Darrah et al., Sci Transl Med,5(186):186ra65 (2013). Despite a similarly important role for PAD2 in RApathogenesis, it is unknown whether this enzyme is also a target of thehumoral response in RA. In this study, we sought to define theprevalence and clinical significance of anti-PAD2 antibodies in patientswith RA by studying a well-defined longitudinal cohort.

SUMMARY OF THE INVENTION

Peptidylarginine deiminases (PAD) 2 and 4 are key enzymes in RApathogenesis due to their ability to generate citrullinated proteins,hallmark targets of autoantibodies in patients with RA. Anti-PAD4autoantibodies have been identified and are associated with severe jointand lung disease. Here, we examined whether anti-PAD2 antibodies werepresent in patients with RA and defined their potential clinicalsignificance.

A PAD2 ELISA assay was established to screen for anti-PAD2 IgG in serafrom RA patients and healthy controls. The clinical and demographiccharacteristics of RA patients were compared according to theiranti-PAD2 antibody status and level. Multivariable models wereconstructed to explore the independent associations of anti-PAD2antibodies with clinical variables. The frequency of anti-PAD2antibodies in RA patients was 18.5%, and the median anti-PAD2 antibodylevel was 75% higher in RA patients compared with control individuals(p=0.0012). Among RA patients, anti-PAD2 antibodies were inverselyassociated with HLA-DRβ1 shared epitope alleles, swollen joint count,and interstitial lung disease (ILD), and were not associated withseropositive disease. After adjusting for relevant confounders,anti-PAD2 antibodies were independently and significantly associatedwith fewer swollen joints, a lower frequency of ILD, and lessprogression of radiographic joint damage. Interestingly, anti-PAD2antibodies did not affect PAD2 enzymatic activity, suggestingalternative mechanism(s) for how these antibodies may contribute to amilder disease phenotype. Anti-PAD2 antibodies represent a novelserologic marker in RA that identifies a genetically and clinicallyunique subset of patients with less severe joint and lung disease.

BRIEF DESCRIPTION OF THE FIGURES

The present invention may be more readily understood by reference to thefollowing figures, wherein:

FIG. 1 provides a graph showing Anti-PAD2 antibodies are found in asubset of patients with RA. Anti-PAD2 antibody arbitrary units (AU) areplotted for each RA patient and healthy control (HC) serum tested. Thecutoff for anti-PAD2 positivity is indicated (---) and was set at3-standard-deviations above the mean of the HC sample excludingstatistical outliers. The median and 95% confidence intervals for eachgroup of sera are shown. The median anti-PAD2 level in patients in theESCAPE RA cohort was compared to the healthy controls and a p<0.05 wasconsidered significant.

FIGS. 2A-2D provide graphs showing anti-PAD2 antibodies are associatedwith fewer swollen joints and less ILD. Baseline mean SJC of 28 (A) or44 (B) joints, mean CT-ILD score (C), and adjusted frequency of ILD (D)according to anti-PAD2 antibody status is shown. (A and B) Model 1 isunadjusted. Model 2 is adjusted for sex, BMI, RA duration, HAQ, biologicuse. Model 3 is additionally adjusted for RF, anti-CCP, anti-PAD3/4, andSE. Model 4 is additionally adjusted for CRP. (D) Adjusted for age, everand current smoking, RF, anti-CCP, anti-PAD3/4, DAS28, and currentbiologic use. (A-D) The average values group 95% confidence intervals,and error bars are shown. A p-value <0.05 was considered significant(*).

FIGS. 3A-3C provide graphs showing anti-PAD2 antibodies are inverselyassociated with progressive joint damage. (A) Mean average SJC for RApatients at all three visits is shown according to their anti-PAD2antibody status. The models are adjusted for the co-variates indicatedin FIG. 2A and B. (B) Yearly change in SHS is plotted versus anti-PAD2units for each patient. (C) Anti-PAD2 antibody level was plotted againstthe frequency of radiographic progression in unadjusted (circles) andadjusted (line) models, and the least squares estimate of theassociation from multivariable linear regression with its associated 95%confidence interval (gray dotted line) is shown. A p-value <0.05 wasconsidered significant (*). AU=arbitrary units

FIGS. 4A and 4B provide images showing Anti-PAD2 antibodies do not alterPAD2 activity in vitro. PAD2 was pre-incubated with IgG from anti-PAD2positive (lanes 3-8) or negative (lanes 9-13) patients, or no IgG(lane 1) prior to incubation with (A) purified human fibrinogen or (B)HEK293 cell lysate in the presence of 1.5 mM CaCl₂ and 1 mM DTT.Immunoblotting was performed using (A) mouse anti-citrullinatedfibrinogen antibody clone 20B2 or (B) mouse anti-peptidyl-citrullineantibody clone F95. Human IgG light chain (IgG-Lc), and a portion of thePonceau-stained membrane (prior to immunoblotting) is shown as a loadingcontrol. (A) The alpha, beta, and gamma isoforms of fibrinogen areindicated. Data is representative of two independent experiments.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which these exemplary embodiments belong. The terminologyused in the description herein is for describing particular exemplaryembodiments only and is not intended to be limiting of the exemplaryembodiments. As used in the specification and the appended claims, thesingular forms “a,” “an,” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

“Treating”, as used herein, means ameliorating the effects of, ordelaying, halting or reversing the progress of a disease or disorder.The word encompasses reducing the severity of a symptom of a disease ordisorder and/or the frequency of a symptom of a disease or disorder. Asubject is successfully “treated” for a disorder characterized byincreased AA levels if the subject shows observable and/or measurablereduction in or absence of one or more signs and symptoms of aparticular disease or condition.

A “subject”, as used therein, can be a human or non-human animal.Non-human animals include, for example, livestock and pets, such asovine, bovine, porcine, canine, feline and murine mammals, as well asreptiles, birds and fish. Preferably, the subject is human.

The language “effective amount” or “therapeutically effective amount”refers to a nontoxic but sufficient amount of the composition used inthe practice of the invention that is effective to provide effectivetreatment in a subject. That result can be reduction and/or alleviationof the signs, symptoms, or causes of a disease or disorder, or any otherdesired alteration of a biological system. An appropriate therapeuticamount in any individual case may be determined by one of ordinary skillin the art using routine experimentation.

The term antibody, as used herein and unless further limited, refers tosingle chain, two-chain, and multi-chain proteins and glycoproteinsbelonging to the classes of polyclonal, monoclonal, chimeric and heteroimmunoglobulins ; it also includes synthetic and genetically engineeredvariants of these immunoglobulins. The term “Antibody fragment” includesFab, Fab′, F(ab′)2, and Fv fragments, as well as any portion of anantibody having specificity toward a desired target epitope or epitopes.

An “autoantibody” (abbreviated “AA”) is an antibody produced by theimmune system of a subject that is directed against one or more of thesubject's own proteins (e.g., PAD2).

Antibodies are grouped into classes, also referred to as isotypes, asdetermined genetically by the constant region. Human constant lightchains are classified as kappa (Cκ) and lambda (Cλ) light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. TheIgG class is the most commonly used for therapeutic purposes. In humansthis class comprises subclasses IgG1, IgG2, IgG3, and IgG4. In mice thisclass comprises subclasses IgG1, IgG2a, IgG2b, IgG3. IgM has subclasses,including, but not limited to, IgM1 and IgM2. IgA has severalsubclasses, including but not limited to IgA1 and IgA2. Thus, “isotype”as used herein is meant any of the classes or subclasses ofimmunoglobulins defined by the chemical and antigenic characteristics oftheir constant regions. The known human immunoglobulin isotypes areIgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE.

The term “Fab” or “Fab region” as used herein includes the polypeptidesthat comprise the VH, CH1, VL, and CL immunoglobulin domains. Fab mayrefer to this region in isolation, or this region in the context of afull length antibody or antibody fragment.

The term “Fc” or “Fc region”, as used herein includes the polypeptidecomprising the constant region of an antibody excluding the firstconstant region immunoglobulin domain. Thus Fc refers to the last twoconstant region immunoglobulin domains of IgA, IgD, and IgG, and thelast three constant region immunoglobulin domains of IgE and IgM, andthe flexible hinge N-terminal to these domains.

Antibody fragments include, but are not limited to, (i) the Fab fragmentconsisting of VL, VH, CL and CH1 domains, including Fab′ and Fab′-SH,(ii) the Fd fragment consisting of the VH and CH1 domains, (iii) the Fvfragment consisting of the VL and VH domains of a single antibody; (iv)the dAb fragment (Ward et al., 1989, Nature 341:544-546) which consistsof a single variable, (v) F(ab′)2 fragments, a bivalent fragmentcomprising two linked Fab fragments (vi) single chain Fv molecules(scFv), wherein a VH domain and a VL domain are linked by a peptidelinker which allows the two domains to associate to form an antigenbinding site (Bird et al., 1988, Science 242:423-426, Huston et al.,1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883), (vii) bispecificsingle chain Fv dimers (PCT/US92/09965), (viii) “diabodies” or“triabodies”, multivalent or multi-specific fragments constructed bygene fusion (Tomlinson et. al., 2000, Methods Enzymol. 326:461-479;WO94/13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A.90:6444-6448) and (ix) scFv genetically fused to the same or a differentantibody (Coloma & Morrison, 1997, Nature Biotechnology 15, 159-163).

The term antigen, as used herein, refers to a molecule or a portion of amolecule capable of being bound by an antibody which is additionallycapable of inducing an animal to produce an antibody capable of bindingto an epitope of that antigen. An antigen can have one or more than oneepitope. The specific reaction referred to above is meant to indicatethat the antigen will react, in a highly selective manner, with itscorresponding antibody and not with the multitude of other antibodieswhich can be evoked by other antigens.

The term epitope, as used herein, refers to that portion of any moleculecapable of being recognized by, and bound by, an antibody. In general,epitopes consist of chemically active surface groupings of molecules,for example, amino acids or sugar side chains, and have specificthree-dimensional structural characteristics as well as specific chargecharacteristics. The epitopes of interest for the present invention areepitopes comprising amino acids.

As used herein, the term “capture probe” refers to a molecule capable ofbinding to a target analyte, e.g., a disease-associated antibody. Oneexample of a capture probe includes antigens that recognize antibodiespresent in a biological sample from patients having or suspected ofhaving a disease, e.g., rheumatoid arthritis.

As used herein, the term “immunoassay” refers to an assay in which anantibody (e.g. anti-PAD2) specifically binds, for example, a captureprobe recognized by or, including an antigen recognized by, the antibody(e.g. PAD2) to provide for the detection and/or quantitation of theantibody. An “immunoassay” can use a particular capture probe to detect,isolate, target, and/or quantify the antibody that specifically binds tothe capture probe. One example of an “immunoassay” includes a captureprobe that contains one or more antigens to detect, isolate, and/orquantify one or more antibodies in a sample.

As used herein, the term “particle based multi-analyte test” (PMAT)refers to an assay that allows simultaneous measurement of two or moreanalytes in a single assay. In some embodiments, a PMAT is a type ofmultiplex assay. For example, a PMAT can use different types ofparticles simultaneously, with each type having immobilized a specificcapture probe for a specific autoantibody on the surface of itsparticles. A PMAT can also include a particle with a plurality ofcapture probes to one or more autoantibodies on the surface of the sameparticle. The particle can include beads, and other small substratefragments.

The term monoclonal antibody, as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variants that mayarise during production of the monoclonal antibody, such variantsgenerally being present in minor amounts. In contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different epitopes, each monoclonal antibody isdirected against a single epitope on the antigen. In addition to theirspecificity, the monoclonal antibodies are advantageous in that they areuncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies.

The term chimeric antibody, as used herein, refers to an antibody whichincludes sequences derived from two different antibodies, whichtypically are of different species. Most typically, chimeric antibodiesinclude human and non-human antibody fragments, generally human constantand non-human variable regions.

The term humanized antibody, as used herein, refers to a type ofchimeric antibody derived from a non-human antibody, and a humanantibody which retains or substantially retains the antigen-bindingproperties of the parent antibody but which is less immunogenic inhumans.

As used herein, a humanized antibody comprises heavy or light chainvariable framework regions that are “the product of” or “derived from” aparticular human germline sequence (human gene) if the variableframework regions of the antibody are obtained from a system that useshuman germline immunoglobulin genes. Such systems include immunizing atransgenic mouse carrying human immunoglobulin genes with the antigen ofinterest or screening a human immunoglobulin gene library displayed onphage with the antigen of interest. A humanized antibody which comprisesa heavy or light chain variable framework region that is “the productof” or “derived from” a human germline immunoglobulin sequence can beidentified as such by comparing the amino acid sequence of the heavy orlight chain variable framework region of the humanized antibody to theamino acid sequences of the heavy or light chain variable frameworkregion of human germline immunoglobulins. A humanized antibody thatcomprises a heavy or light chain variable framework region that is “theproduct of” a particular human germline immunoglobulin sequence has aheavy or light chain variable framework region which is 100% identicalin amino acid sequence to the heavy or light chain variable frameworkregion of the particular human germline immunoglobulin sequence. Ahumanized antibody that comprises a heavy or light chain variableframework region that is “derived from” a particular human germlineimmunoglobulin sequence may contain amino acid differences as comparedto the heavy or light chain variable framework region of the particulargermline sequence, due to, for example, naturally-occurring somaticmutations or intentional introduction of site-directed mutation.However, a selected humanized antibody typically is at least 90%identical in amino acid sequence of the heavy or light chain variableframework region to an amino acid sequence encoded by the heavy or lightchain variable framework region of a human germline immunoglobulin geneand contains amino acid residues that identify the humanized antibody asbeing derived from human when compared to the germline immunoglobulinamino acid sequences of other species (e.g., murine germline sequences).

As used herein, the phrase “specifically binds” refers to antibodybinding to a target structure, wherein the antibody binds a targetstructure, or subunit thereof, but does not bind to a biologicalmolecule that is not a target structure. Antibodies that specificallybind to a target structure, or subunit thereof, do not cross-react withbiological molecules that are outside the target structure family Anantibody specific for PAD2 can be an antibody or antibody fragmentcapable of binding to that specific protein with a specific affinity ofbetween 10⁻⁸ M and 10⁻¹¹ M. In some embodiments, an antibody or antibodyfragment binds to a selected antigen with a specific affinity of greaterthan 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, or 10⁻¹¹ M, between 10⁻⁸ M-10⁻¹¹M, 10⁻⁹ M-10⁻¹⁰ M, and 10⁻¹⁰ M-10⁻¹¹ M. In a preferred aspect, specificactivity is measured using a competitive binding assay as set forth inAusubel FM, (1994). Current Protocols in Molecular Biology. Chichester:John Wiley and Sons (“Ausubel”), which is incorporated herein byreference.

Obtaining a Prognosis for a Subject having Rheumatoid Arthritis

The present invention provides for prognostic (or predictive) assays fordetermining the risk that a subject will develop a more severe form ofrheumatoid arthritis. Such assays can be used for prognostic orpredictive purpose, for example to select appropriate therapeutic orprophylactic compounds for a subject based on the level of AA (e.g.,anti-PAD2 antibodies) in a sample obtained from the subject. The assaysalso can be used for disease diagnosis, including the severity of thedisease, and/or monitoring the disease, including monitoring theresponse to a treatment. The methods described herein can also be usedto determine the levels of such AAs in subjects to aid in predicting theresponse of such subjects to medication.

One aspect of the invention provides a method for obtaining a prognosisfor a subject having rheumatoid arthritis (RA) comprising: a) obtaininga sample from a subject having RA; b) providing a substrate having afirst capture probe bound thereto, wherein the capture probe comprisespeptidyl arginine deiminase 2 (PAD2) protein or a portion or fragmentthereof which comprises an antigen recognized by autoantibodies presentin subjects suffering from RA; c) contacting the substrate having thecapture probe bound thereto with the sample from the subject; d)measuring the amount of a complex of the capture probe and theautoantibodies formed in step c); e) providing a reference level sample;f) comparing the amount of a complex of the capture probe and theautoantibodies formed from the subject having RA to the amount of acomplex of the capture probe and the antibodies formed from thereference level sample; and g) identifying the subject as having a lowerrisk of developing a severe form of RA when the amount of a complex ofthe capture probe and the autoantibodies formed from the subject havingRA is increased compared to the amount of a complex of the capture probeand the autoantibodies formed from the reference level sample. In someembodiments, the invention provides a method for obtaining a diseasediagnosis for RA, including, for example, diagnosis of a subjectsuspected of having a less severe form of RA using the methods providedherein. In some embodiments, the invention provides a method formonitoring, including therapeutic monitoring, a subject with RA, usingthe methods provided herein. In some embodiments, the invention providesa method for monitoring disease progression of a subject with RA, usingthe methods provided herein.

The capture probe includes peptidyl arginine deiminase 2 (PAD2) proteinor a portion or fragment thereof. PAD2 is one of five PAD enzymesencoded by humans, and appears to drive citrullination of self-antigensin rheumatoid arthritis. PAD enzymes (e.g., PAD2) are calcium-dependentenzymes. The structure of PAD2 has been characterized. See Slade et al.,ACS Chem Biol. 10(4), 1043-53 (2015). Portions or fragments of PAD2 canalso be used. Portions or fragments of PAD2 include peptides comprisingamino acid sequences sufficiently homologous to or derived from theamino acid sequence of a PAD2 protein, but which include less aminoacids than a full length PAD2 protein. A portion or fragment of PAD2 canbe a polypeptide which is, for example, 10, 25, 50, 100, 200, 300, 400,500, or more amino acids in length. The portion or fragment of PAD2should include at least one epitope binding by anti-PAD2 antibodies.

The evaluation of PAD2 autoantibody level can be carried out using avariety of different types of assays. In some embodiments, the method isan enzyme immunoassay method. In other embodiments, the method is aradioimmunoassay method. In further embodiments, the method is animmunoblotting method. In another embodiment, the method is achemiluminescent immunoassay (CIA). In yet a further embodiment, themethod is a particle based multianalyte test (PMAT).

Methods and protocols for conducting immunoassays and biophysicalprotein-interaction assays are well known in the art. See, e.g., WildD., The Immunoassay Handbook, Elsevier Science, 4^(th) Edition (2013);Fu H., Protein-Protein Interactions, Humana Press, 4^(th) Edition(2004). For example, a chemiluminescent immunoassay (CIA) is animmunoassay technique where the label, for example, the “indicator” ofthe analytic reaction, is a luminescent molecule. In general,luminescence is the emission of visible or near-visible (λ=300-800 nm)radiation which is generated when an electron transitions from anexcited state to ground state. The resultant potential energy in theatom gets released in the form of light.

A PMAT, allows simultaneous measurement of two or more analytes in asingle assay. For example, in PMAT, different types of particles areused simultaneously, with each type having immobilized a specificbinding partner for a specific molecule species on the surface of itsparticles. In a solution, the analyte molecules to be detected are boundto their binding partners on the corresponding particle type. The bondsare then detected optically through the addition of a secondary markerthat marks all particle-bound analyte molecules of the PMAT assay. APMAT can be performed using a variety of formats known in the art, suchas flow cytometry, a capture sandwich immunoassay, or a competitiveimmunoassay. For example, using a dual-laser flow-based detectioninstrument, the binding of analyte fractions, such as autoantibodies,can be detected through the fluorescence of the secondary marker. Insome embodiments, the PMAT particle is a bead.

In effecting an enzymatic, fluorescence, chemical (e.g.chemiluminescence), colormetric or PMAT immunoassay, the level of PAD2autoantibodies is determined by providing a substrate having a firstcapture probe bound thereto, wherein the capture probe comprisespeptidyl arginine deiminase 2 (PAD2) protein or a portion or fragmentthereof which comprises an antigen recognized by autoantibodies presentin the serum of subjects suffering from RA. The substrate is a solidphase; examples include beads made of polystyrene, glass, etc.,microplates and the like. When the PAD2 is adsorbed on the solid phase,there may preferably be employed a biological sample including PAD2having a PAD2 concentration of 0.1 μg/ml or more. It is preferred thatthe PAD2 concentration be predetermined taking into consideration thesensitivity in the assay system. A preferred concentration is about 1 to10 μg/ml.

As used herein, the term “substrate” refers to any surface capable ofhaving capture probes bound thereto. Such surfaces include, but are notlimited to, glass, metal, plastic, or materials coated with a functionalgroup designed for binding of capture probes or analytes. Substratesalso may be referred to as slides.

The capture probe can be bound to the substrate using a binding reagent,or in other embodiments, it can be non-specifically adsorbed to thesubstrate surface. A variety of binding reagents for adhering proteinsto a substrate are known to those skilled in the art. For example,antibodies specific to the protein can be used as binding reagents.Aptamers and various chemical reagents (e.g., coupling agents, such asdialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotizedbenzidine) can also be used as binding reagents.

The PAD protein or fragment thereof thus adsorbed on the solid phase isthen reacted with a biological sample (e.g., serum or plasma) of thesubject. It is preferred that the sample be diluted with, for example, asodium phosphate buffer containing a surfactant 10 to 100 times. Thereaction was effected at 4° C. to 40° C., for 30 minutes to overnight,for example, at 4° C., overnight or at 37° C. for 30 minutes whilepreventing the evaporation of the reaction mixture. Then, the resultingmixture is reacted with a solution of an enzyme-labeled anti-human IgGantibody having an appropriate antibody concentration at 4° C. to 40° C.for 30 minutes to overnight. Thus, only the anti-PAD2 autoantibody inthe sample is caught on the PAD2 protein adsorbed on the solid phase.The immobilized anti-PAD2 autoantibody can then be identified bylabeling it with a labeled molecule that specifically binds to theanti-PAD2 autoantibody. For example, enzyme-labeled anti-human IgGantibody can be used to identify the bound anti-PAD2 autoantibody.

The enzymatic, fluorescence, chemical (e.g. chemiluminescence),colormetric, PMAT, or similar activity revealed by the labeled antibodythus bonded corresponds to the amount of autoantibody immobilized by thecapture probe. Examples of suitable enzymes include alkaline phosphataseand horseradish peroxidase. Accordingly, the amount of anti-PAD2antibody in the sample can be determined by measuring the enzymeactivity. Alternately, other labels can be used instead of enzymeactivity. Examples of other types of label include a radioisotope, afluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin(PE)), chemiluminescence, and element particles (e.g., gold particles).

Methods for measuring the amount or presence of an antibody-PAD2 complexinclude, for example, detection of fluorescence, luminescence,chemiluminescence, absorbance, reflectance, transmittance, birefringenceor refractive index (e.g., surface plasmon resonance, ellipsometry, aresonant mirror method, a gating coupler waveguide method orinterferometry). Optical methods include microscopy (both confocal andnon-confocal), imaging methods and non-imaging methods. Electrochemicalmethods include voltammetry and amperometry methods. Radio frequencymethods include multipolar resonance spectroscopy.

As used herein, the term “sample” means sample material derived from orcontacted by living cells. The term “sample” is intended to includetissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject. Biological samplesinclude, e.g., but are not limited to, whole blood, plasma, serum,semen, cell lysates, saliva, tears, urine, fecal material, sweat,buccal, skin, synovial fluid, cerebrospinal fluid, and hair. Biologicalsamples can also be obtained from biopsies of internal organs.Preferably, the biological sample is a biological fluid includingautoantibodies. Biological samples can be obtained from subjects fordiagnosis, prognosis, monitoring, or a combination thereof, or researchor can be obtained from un-diseased individuals, as controls or forbasic research. Biological samples can be obtained by any known meansincluding needle stick, needle biopsy, swab, and the like.

A biological sample may be fresh or stored (e.g. blood or blood fractionstored in a blood bank). Samples can be stored for varying amounts oftime, such as being stored for an hour, a day, a week, a month, or morethan a month. The biological sample may be a bodily fluid expresslyobtained for the assays of this invention or a bodily fluid obtained foranother purpose which can be sub-sampled for the assays of thisinvention.

As used herein, the term “reference level” is intended to mean a controllevel of a biomarker, e.g., disease-associated AA, used to evaluate atest level of the biomarker (e.g., PAD2 autoantibody) in a sample froman individual. A reference level can be a normal reference level in asample from a normal subject or a disease reference level from adisease-state subject. A normal reference level is an amount ofexpression of a biomarker in a non-diseased subject or subjects. Adisease-state reference level is an amount of expression of a biomarkerin a subject with a positive diagnosis for the disease or condition. Areference level also can be a stage-specific reference level. Astage-specific reference level refers to a level of a biomarkercharacteristic of a given stage of progression of a disease orcondition.

A subject having a decreased or increased risk of developing a severeform of rheumatoid arthritis has a lower or higher (respectively)percentage chance of developing a severe form of arthritis in comparisonwith the average risk that a subject having rheumatoid arthritis willdevelop a severe form of rheumatoid arthritis. For example, a subjecthaving an increased risk of developing a severe form of RA can have a5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%,150%, 200%, or 300% or more higher chance of developing a severe form ofRA in comparison with the risk that an average subject having RA willdevelop a severe form of RA.

The method can also include the step of providing a report indicatingthe subject has a lower risk of developing a severe form of RA, or is inneed of therapy suitable to prevent the development of a severe RA. Forexample, the apparatus for carrying out the method can include aprocessor coupled to the protein detector and adapted to quantify thedata representing the signals from the detector, and adapted to performthe multivariate statistical analysis, compare the output value to thefirst reference value and the second reference value, and calculate therisk score; and an output display coupled to the processor andconfigured to report the risk score.

The invention includes the step of providing a prognosis for a subjecthaving rheumatoid arthritis (RA) which includes identifying the subjectas having a lower risk of developing a severe form of RA when the amountof a complex of the capture probe and the autoantibodies formed from thesubject having RA is increased compared to the amount of a complex ofthe capture probe and the autoantibodies formed from the reference levelsample. In some embodiments, the invention includes the step ofdiagnosing a subject suspected of having RA. The invention also includesdiagnosing a less severe form of RA. For example, a less severe form ofRA can be diagnosed when the amount of a complex of the capture probeand the autoantibodies formed from the subject having RA is increasedcompared to the amount of a complex of the capture probe and theautoantibodies formed from the reference level sample. In someembodiments, the invention includes the step of monitoring a subjectwith RA, which includes monitoring the amount of a complex of thecapture probe and the autoantibodies formed from the subject having RAcompared to the amount of a complex of the capture probe and theautoantibodies formed from the same subject at a previous time. In someembodiments, the monitoring can include monitoring the therapeuticresponse of a patient being treated for RA. In some embodiments, themonitoring can include monitoring disease progression of RA. Rheumatoidarthritis (RA) is a type of connective tissue disease that is along-term autoimmune disorder characterized by symmetric immune-mediateddamage to the joints, resulting in warm, swollen, and painful joints,and is believed to result from a combination of genetic andenvironmental factors. RA can be diagnosed using imaging, such as x-ray,MRI, or ultrasound imaging. RA can also be diagnosed and/or monitoredusing blood tests for the presence of rheumatoid factor andanti-citrullinated protein antibodies. The inventors have alsopreviously described evaluating anti-PAD3/PAD4 cross-reactive antibodylevels to predict severe erosive RA as well as the presence ofinterstitial lung disease in a subject. See U.S. Pat. No. 9,417,247.

The lung is a common site of complications of systemic connective tissuediseases, such as RA and other connective tissue diseases. Lunginvolvement can present as interstitial lung disease (ILD). Connectivetissue disease associated with interstitial lung disease (CT-ILD), alsoknown as interstitial pneuomonia with autoimmune features (IPAF), is aserious pulmonary complication associated with connective tissuediseases, and IPAF can indicate a more severe type of connective tissuedisease. For example, severe RA involves a higher level of articulardamage, and/or RA-associated conditions, such as interstitial lungdisease (RA-ILD). Additional connective tissue diseases associated withthe development of IPAF include, but are not limited to, systemicsclerosis (SSc), scleroderma, sarcoidosis, poly-/dermatomyositis(PM/DM), Sjogren's syndrome (SjS), systemic lupus erythematosus (SLE),and undifferentiated (UCTD) as well as mixed connective tissue disease(MCTD). The manifestation of particular connective tissue diseases, suchas RA, to include features of ILD are indicators of severe forms of thedisease. Thus, in some embodiments, provided herein are methods for thediagnosis, including disease severity, prognosis, and/or monitoring forvarious forms of IPAF such as those exemplified above and RA-ILD. Severerheumatoid arthritis is also referred to as erosive RA, and can beidentified using an X-ray evaluation of the subject which shows evidenceof cartilage and/or bone destruction.

Treatment of subjects having RA is often more aggressive than necessarybecause subjects having an increased risk of developing severe RA cannotbe identified. The present invention provides a method of avoiding suchexpensive and unnecessary treatment in subjects who have a lower risk ofdeveloping severe RA. Rheumatoid arthritis is typically treated with adisease modifying anti-rheumatic drug (DMARD) such as methotrexate.However, if the disease continues to worsen, or does not improve in 3-6months, the treating physician may initiation combination therapy byadding additional non-biologic DMARDs or switching the patient to asecond-line drug like a biological medication (e.g., a TNF-α inhibitor).However, because destruction of bone and damage to the lungs isirreversible, physicians may immediately provide more aggressivetreatment such as combination therapy to help assure that severe RA doesnot develop.

Methods of Treating Rheumatoid Arthritis

Another aspect of the invention provides a method of treating rheumatoidarthritis (RA) in a subject by administering to the subject atherapeutically effective amount of an antibody or a fragment thereofthat specifically binds to peptidyl arginine deiminase 2 (PAD2) protein.Treatment of a subject having RA with anti-PAD2 protein can help avoidprogression of the RA to a more severe form of RA (i.e., erosive RA). Insome embodiments, the invention provides a method of treating IPAF,and/or RA-ILD in a subject by administering to the subject atherapeutically effective amount of an antibody or a fragment thereofthat specifically binds to peptidyl arginine deiminase 2 (PAD2) protein.

The anti-PAD2 antibody can be any type of immunoglobulin that is knownin the art. For instance, the antibody can be of any isotype, e.g., IgA,IgD, IgE, IgG, IgM, etc. The antibody can be monoclonal or polyclonal.The antibody can be a naturally-occurring antibody, e.g., an antibodyisolated and/or purified from a mammal, e.g., mouse, rabbit, goat,horse, chicken, hamster, human, etc. Alternatively, the antibody can bea genetically-engineered antibody, e.g., a humanized antibody or achimeric antibody. The antibody can be in monomeric or polymeric form.

Methods of testing antibodies for the ability to bind to antigens (e.g.,PAD2 antigens) are known in the art and include any antibody-antigenbinding assay, such as, for example, radioimmunoassay (RIA), ELISA,Western blot, immunoprecipitation, and competitive inhibition assays(see, e.g., Janeway et al., infra, and U.S. Patent ApplicationPublication No. 2002/0197266 A1).

Suitable methods of making antibodies to PAD2 are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Kohler andMilstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.),Antibodies: A Laboratory Manual, CSH Press (1988), and C. A. Janeway etal. (eds), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y.(2001)). Alternatively, other methods, such as EBV-hybridoma methods(Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), andRoder et al., Methods Enzymol., 121, 140-67 (1986)), and bacteriophagevector expression systems (see, e.g., Huse et al., Science, 246, 1275-81(1989)) are known in the art. Further, methods of producing antibodiesin non-human animals are described in, e.g., U.S. Pat. Nos. 5,545,806,5,569,825, and 5,714,352, and U.S. Patent Application Publication No.2002/0197266 A1).

Phage display furthermore can be used to generate antibodies. In thisregard, phage libraries encoding antigen-binding variable (V) domains ofantibodies can be generated using standard molecular biology andrecombinant DNA techniques (see, e.g., Sambrook et al. (eds.), MolecularCloning, A Laboratory Manual, 3rd Edition, Cold Spring Harbor LaboratoryPress, New York (2001)). Phage encoding a variable region with thedesired specificity are selected for specific binding to the desiredantigen, and a complete or partial antibody is reconstituted comprisingthe selected variable domain. Nucleic acid sequences encoding thereconstituted antibody are introduced into a suitable cell line, such asa myeloma cell used for hybridoma production, such that antibodieshaving the characteristics of monoclonal antibodies are secreted by thecell (see U.S. Pat. No. 6,265,150).

Antibodies can be produced by transgenic mice that are transgenic forspecific heavy and light chain immunoglobulin genes. Such methods areknown in the art and described in, for example U.S. Pat. Nos. 5,545,806and 5,569,825, and Janeway et al., supra.

Humanized antibodies can be produced using synthetic and/or recombinantnucleic acids to prepare genes (e.g., cDNA) encoding the desiredhumanized chain. For example, nucleic acid (e.g., DNA) sequences codingfor humanized variable regions can be constructed using PCR mutagenesismethods to alter DNA sequences encoding a human or humanized chain, suchas a DNA template from a previously humanized variable region (see e.g.,Kamman, M., et al., Nucl. Acids Res., 17: 5404 (1989)); Sato, K., etal., Cancer Research, 53: 851-856 (1993); Daugherty, B. L. et al.,Nucleic Acids Res., 19(9): 2471-2476 (1991); and Lewis, A. P. and J. S.Crowe, Gene, 101: 297-302 (1991)). Using these or other suitablemethods, variants can also be readily produced. In one embodiment,cloned variable regions can be mutagenized, and sequences encodingvariants with the desired specificity can be selected (e.g., from aphage library; see e.g., Krebber et al., U.S. Pat. No. 5,514,548;Hoogenboom et al., WO 93/06213).

The anti-PAD2 antibody is administered in an effective amount whichinhibits the activity of PAD2. For therapy, an effective amount will besufficient to achieve the desired therapeutic (including prophylactic)effect (such as an amount sufficient to inhibit PAD2 activity). Theantibody can be administered in a single dose or multiple doses. Thedosage can be determined by methods known in the art and can bedependent, for example, upon the individual's age, sensitivity,tolerance and overall well-being. Suitable dosages for antibodies can befrom about 0.1 mg/kg body weight to about 10.0 mg/kg body weight pertreatment.

According to the method, the antibody (e.g., humanized immunoglobulin)can be administered to an individual (e.g., a human) alone or inconjunction with another agent. A humanized immunoglobulin can beadministered before, along with or subsequent to administration of theadditional agent. Thus, the invention includes pharmaceuticalcompositions comprising an anti-PAD2 antibody or fragment thereof of theinvention and a suitable carrier. In one embodiment, more than onedifferent anti-PAD2 antibody is administered. In another embodiment, anadditional pharmacologically active ingredient (e.g., an agent suitablefor treating rheumatoid arthritis, such as methotrexate,hydroxychloroquine, sulfasalazine, leflunomide, TNF-α inhibitors(certolizumab, infliximab and etanercept), abatacept, anakinra,rituximab and tocilizumab can be administered in conjunction with ananti-PAD2 antibody of the present invention. A variety of routes ofadministration are possible, including, but not necessarily limited to,parenteral (e.g., intravenous, intraarterial, intramuscular,subcutaneous injection), oral (e.g., dietary), topical, inhalation(e.g., intrabronchial, intranasal or oral inhalation, intranasal drops),or rectal, depending on the disease or condition to be treated.Parenteral administration is a preferred mode of administration.

Formulation will vary according to the route of administration selected(e.g., solution, emulsion). An appropriate composition comprising theanti-PAD2 antibody to be administered can be prepared in aphysiologically acceptable vehicle or carrier. For solutions oremulsions, suitable carriers include, for example, aqueous oralcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles can include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils. Intravenous vehicles can include variousadditives, preservatives, or fluid, nutrient or electrolyte replenishers(See, generally, Remington's Pharmaceutical Sciences, 17th Edition, MackPublishing Co., PA, 1985). For inhalation, the compound can besolubilized and loaded into a suitable dispenser for administration(e.g., an atomizer, nebulizer or pressurized aerosol dispenser).

The antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be, for example, weekly, monthly, every threemonths or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of antibody to the target antigen in the patient.In some methods, dosage is adjusted to achieve a plasma antibodyconcentration of about 1-1000 μg/ml and in some methods about 25-300μg/ml. Alternatively, antibody can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of theantibody in the patient. The dosage and frequency of administration canvary depending on whether the treatment is prophylactic or therapeutic.In prophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated.

The selected dosage level will depend upon a variety of pharmacokineticfactors including the activity of the particular compositions of thepresent invention employed, the route of administration, the time ofadministration, the rate of excretion of the particular antibody beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

In some embodiments, the anti-PAD2 antibody is a humanized antibody.Humanized antibodies are preferable for use in human subjects, in orderto avoid generating an immune response against the antibodiesthemselves. Examples of humanized antibodies include those selected fromthe group of antibodies consisting of Fab2, Fab4, and Fab6, or variantsthereof including only conservative sequence modifications.

Kits

In accordance with another embodiment, the present invention providesone or more kits for providing a prognosis, diagnosis, including theseverity of the disease, and/or monitoring for a subject havingrheumatoid arthritis (RA), or for treating a subject having rheumatoidarthritis. In some embodiments, the present invention provides one ormore kits for providing a prognosis, diagnosis, and/or monitoring for asubject having IPAF, RA-ILD, or for treating a subject having IPAF, orRA-ILD. The kits components of the kits will vary depending on whetherthey are intended for prognosis, diagnosis, monitoring, or treatment.Kits for prognosis, diagnosis, or monitoring comprise a substrateincluding a capture probe, wherein the capture probe comprises peptidylarginine deiminase 2 (PAD2) protein, or a portion or fragment thereof.The kit also includes reagents, buffers, and the like for carrying outan immunoassay (e.g., ELISA), which are known to those of ordinary skillin the art. Kits for treatment include anti-PAD2 antibodies, oreffective fragments thereof, together with a pharmaceutically acceptablecarrier for administration of the antibodies. The antibody or fragmentthereof that specifically binds to PAD2 can be any of the antibodiesdescribed herein. For example, in some embodiments the antibody isselected from the group of antibodies consisting of Fab2, Fab4, andFab6, or variants thereof including only conservative sequencemodifications

Kits according to the present invention are assemblies of reagents fortesting antibody binding, or administering antibodies. They aretypically in a package which contains all elements, optionally includinginstructions. Instructions may be in any form, including paper ordigital. The instructions may be on the inside or the outside of thepackage. The instructions may be in the form of an internet addresswhich provides the detailed manipulative or analytic techniques. Thepackage may be divided so that components are not mixed until desired.

Components of the kits of the present invention may be in differentphysical states. For example, some components may be lyophilized andsome in aqueous solution. Some may be frozen. Individual components maybe separately packaged within the kit. Other useful tools for performingthe methods of the invention or associated testing, therapy, orcalibration may also be included in the kits, including buffers,enzymes, chemiluminesce reagents, PMAT reagents, gels, plates,detectable labels, vessels, etc. Kits may-include tools for collectingsuitable samples, such as tools for collecting oral swabs, oralbiopsies, and endoscopes.

An example has been included to more clearly describe a particularembodiment of the invention and its associated cost and operationaladvantages. However, there are a wide variety of other embodimentswithin the scope of the present invention, which should not be limitedto the particular examples provided herein.

EXAMPLE

Autoantibodies to Peptidylarginine Deiminase 2 are Associated with LessSevere Disease in Rheumatoid Arthritis

In this study, we found that anti-PAD2 antibodies are present in asubset of patients with RA who lack the traditional risk factorsassociated with severe disease, such as ACPAs and SE. Instead, they havea unique set of demographic and clinical characteristics including lessprogressive articular damage and lower risk of ILD. These findings haveimportant implications in the ability to identify clinically informativepatient subgroups to aid in disease prognosis and selection ofappropriate therapeutic agents.

Methods

Human Subjects

Convenience sera from 37 healthy controls and 42 patients with RA fromthe Johns Hopkins Arthritis Center were used as a discovery sample foranti-PAD2 antibodies. Sera from 184 RA patients from the Evaluation ofSubclinical Cardiovascular Disease and Predictors of Events inRheumatoid Arthritis (ESCAPE RA) cohort were then screened for thepresence of PAD2 autoantibodies by ELISA. All patients in ESCAPE RA metthe American Rheumatism Association 1987 revised criteria for theclassification of RA (Arnett et al., Arthritis Rheum, 31(3):315-324(1988)) and have been extensively described previously. Giles et al.,Ann Rheum Dis, 73(8):1487-1494 (2014). In brief, baseline demographicdata and medication use were captured by questionnaire; anti-CCP, RF,and anti-PAD3/4 antibodies were measured as previously reported (Darrahet al., ibid); and clinical features were assessed by clinicalexamination. Radiographs of the hands and feet, obtained at baseline anda follow-up visit occurring 39±4 months after baseline, were scoredaccording to the Sharp-van der Heijde method by an experienced readerblinded to clinical characteristics. The change in Sharp-van der Heijdescore (SHS) between the two visits was calculated. The number of swollenjoints was recorded at each study visit and the average mean swollenjoint count (SJC) throughout the duration of the study was determinedusing an area-under-the-curve calculation. Participants underwentmultidetector row computed tomography (MDCT) of the chest at thebaseline visit, and the presence and extent of ILD was scored by anexperienced pulmonary radiologist as previously described (Giles et al.,ibid). All samples were obtained under informed written consent approvedby the Johns Hopkins Institutional Review Board.

PAD2 Protein Purification

Recombinant human PAD2 was expressed from the pET28a vector, generatinga protein containing both N-terminal 6×Histidine and T7 tags. Theprotein was purified using a Ni-NTA agarose column according tomanufacturer's instructions (Qiagen). Following PAD2 purification, the6xHis tag was removed by cleavage with thrombin.

Anti-PAD2 ELISA

High-binding EIA plates (Costar) were coated overnight with 200 ng/wellof PAD2 in phosphate buffered saline pH 7.4 (PBS) or PBS alone. Plateswere blocked with 3% non-fat milk. Patient sera were diluted 1:250 in 1%milk/PBS/0.05% tween-20 and assayed in duplicate. A known positivepatient serum was serially diluted and included as a standard on eachplate. Anti-PAD2 units were assigned with the highest standardrepresenting 10 anti-PAD2 arbitrary units (AU). Anti-PAD2 antibodybinding was detected using a horseradish peroxidase-conjugatedanti-human IgG secondary antibody (Jackson Immunoresearch) diluted1:7500 in PBS/0.05% Tween-20. SureBlue TMB peroxidase substrate (KPL)was added to visualize antibody binding and an equal volume of 1Mhydrochloric acid was added to stop the colorimetric reaction, beforedetermining the absorbance at 450 nm with a 560 nm reference using aPerkin Elmer Victor 3 plate reader. The 4-point standard curve formedfrom the serially diluted positive control sera was used to calculateanti-PAD2 AU based on the average absorbance values for each unknownserum sample using WorkOut software. The background values observed forPBS-coated wells were subtracted from the corresponding PAD2-coatedwells for each sample. The threshold for positivity for each cohort wasset at three standard deviations above the mean of the correspondinghealthy control sera, minus statistical outliers. Statistical outlierswere defined as healthy control sera with values 1.5× IQR above thethird quartile or below the first quartile. Data from all individualsscreened is included in FIG. 1.

In Vitro Citrullination Assay

IgG was purified from the serum of 6 anti-PAD4 positive and 5 anti-PAD4negative patients with RA using the Melon Gel IgG Spin Purification Kit(Thermo). Recombinant human PAD2 (200 nM) was pre-incubated with thepurified IgG (1.5 uM) for 45 min at 4° C. in 100 mM Tris-HCl (pH 7.4).Following incubation, plasminogen-depleted fibrinogen (1 μM) (Millipore#341578) or HEK293 cell lysate in NP40 lysis buffer (250 mg/ml) wasadded to the PAD2/IgG mixtures and incubated at 37° C. for 90 mM in thepresence of 1.5 mM CaCl₂ and 1 mM DTT. The citrullination reaction wasstopped directly by adding SDS sample buffer and boiling at 95° C. for 5mins. Denatured proteins were separation by electrophoresis, transferredto nitrocellulose (Ponceau stain used as a loading control), andimmunoblotted using respective antibodies. Mouseanti-peptidyl-citrulline antibody (Millipore, clone F95) was used toassess citrullination of substrates in the HEK293 lysate, and mouseanti-citrullinated fibrinogen (Modiquest, clone 20B2) was used to detectcitrullinated fibrinogen. The blots were incubated with primaryantibodies at dilution of 1:1000 overnight, followed by incubation witha goat anti-mouse secondary antibody at 1:10000 (anti-IgM isotype forF95) for 1 hour at room temperature. Antibody binding was detected usingWest Pico ECL reagent (Pierce) and chemiluminescence was visualized byautoradiography.

Statistical Analyses

The difference in the median anti-PAD2 AU of the RA patients comparedwith healthy controls was determined using a Mann-Whitney rank sum test,and the difference in anti-PAD2 positivity was determined using a 2×2contingency table with Fisher's exact test. For analysis of demographicand clinical variables, RA patients were grouped according to thepresence or absence of anti-PAD2 antibodies, and characteristics werecompared using Intercooled STATA12. Student's t-tests were used forgroup-wise comparisons of normally distributed continuous variables; theKruskal-Wallis test was used for group-wise comparisons of non-normallydistributed variables; and Chi-squared or two-sided Fisher's exacttests, as appropriate, were used for group-wise comparisons ofcategorical variables. We also explored the associations of anti-PAD2level with patient characteristics. For continuous characteristics,Spearman correlations coefficients were calculated. For dichotomouscharacteristics, median (IQR) anti-PAD2 levels were calculated for thosewith and without the dichotomous characteristics of interest, andcompared using the Kruskal-Wallis test. We explored the independentassociation of anti-PAD2 level with radiographic progression and thefrequency of radiographic ILD using multivariable ordinary logisticregression, adjusting for covariates associated with the outcomes ofinterest and anti-PAD2 level at the p<0.20 level in univariate modeling.A similar modeling strategy was used in the context of multivariablelinear regression to explore the association of anti-PAD2 with baselineand average swollen joint counts. A two-tailed alpha=0.05 was usedthroughout.

Results

PAD2 is a Target of Autoantibodies in Patients with RA

To define whether IgG antibodies to PAD2 are present in patients withRA, we established a PAD2 ELISA assay and initially screened conveniencesera from RA patients (n=46) and healthy controls (n=37). Using a cutoffof three standard deviations above the mean of the healthy controls, 10(21.7%) of the RA patients and 2 (5.4%) of the controls were positivefor anti-PAD2 antibodies (FIG. 1). To address the prevalence andclinical significance of anti-PAD2 antibodies in a well-defined cohortof patients with RA, sera from the longitudinal ESCAPE RA cohort (n=184)were analyzed. Anti-PAD2 antibodies were found in 18.5% of patients inthe ESCAPE-RA cohort, and the median anti-PAD2 antibody level wassignificantly higher in the RA group compared with healthy controls(1.68 vs. 0.96 AU, respectively; p=0.0012).

Anti-PAD2 Antibodies Identify a Genetically Distinct RA Patient Subset

To determine if anti-PAD2 antibodies were associated with specificserologic, genetic, or demographic characteristics within the RApopulation, patients were grouped according to their anti-PAD2 antibodystatus and variables collected at their baseline visit were compared(summarized in Table 1). The median anti-PAD2 level by clinicalcharacteristic is also reported in Table 2. The analysis revealed that82% of RA patients with anti-PAD2 antibodies were female, asignificantly higher proportion than present in the anti-PAD2 negativegroup (p=0.003) (Table 1). This corresponded to a 43% higher anti-PAD2antibody level in female vs. male RA patients (p=0.013) (Table 2).Anti-PAD2 antibody levels were also positively associated with age(r=0.183) (Table 2). Importantly, individuals with anti-PAD2 antibodieswere significantly less likely to have SE alleles compared withanti-PAD2 negative patients (53% vs. 74%, respectively) (Table 1),corresponding to a 36% lower anti-PAD2 level in patients with SE alleles(p=0.02). Anti-PAD2 antibodies were not associated with other knownserologies including anti-CCP, RF, or anti-PAD3/4 antibodies (Table 1).

Anti-PAD2 Antibodies Identify Patients with Less Severe Baseline JointInflammation and Lung Involvement.

Analysis of baseline clinical variables revealed that patients withanti-PAD2 antibodies had a significantly lower median swollen jointcount (SJC) at baseline, compared to those without anti-PAD2 (2 vs. 4joints, respectively; p=0.049) (Table 1). This was observed whether 28or 44 joints were evaluated (FIG. 2A and B). The presence ofradiographic ILD was also less prevalent in anti-PAD2 antibody positiveversus negative patients (18% vs. 36%, respectively; p=0.06) (Table 1),and antibody levels were 21% lower in patients who had ILD (p=0.039)(Table 2). It is important to note that the average ILD score amongpatients in the ESCAPE RA was low, since patients were classified ashaving radiographic ILD if any features of ILD were seen on MDCT,irrespective of clinical symptoms. Giles et al., PLoS One, 9(6):e98794(2014). The lower frequency of ILD associated with anti-PAD2 wasprimarily driven by a difference in the finding of a dominant pattern ofground glass opacification (GGO) on CT. While 20 of the 131 anti-PAD2negative patients (15%) had GGO as the predominant ILD pattern, 0% ofthe anti-PAD2 positive individuals (p=0.026) had this radiographicfinding. In contrast, 31 of the 131 patients (24%) without anti-PAD2antibodies had reticulation, honeycombing, or traction bronchiectasis astheir predominant ILD pattern, compared with 5 of the 27 (19%) patientswith anti-PAD2 antibodies (p=0.80). As such, the CT-ILD score was 66%lower in patients with anti-PAD2 antibodies compared to those who wereanti-PAD2 negative (p=0.021, FIG. 2C).

It is notable that anti-PAD2 antibody levels were 25% higher in peoplewho were receiving treatment with biologic disease modifyingantirheumatic drugs (DMARDs) at the time of the study (Table 2,p=0.039), but the percentage of patients on biological DMARDs did notdiffer between the anti-PAD2 positive and negative groups (Table 1,p=0.17). In addition, anti-PAD2 antibody levels did not significantlydiffer based on treatment with non-biologic medications orglucocorticoids (Table 2).

Multivariable models were developed to define the independentcontribution of anti-PAD2 antibodies to baseline joint counts and CT-ILDfeatures. After adjusting for sex, body-mass index (BMI), diseaseduration, health assessment questionnaire (HAQ), biological DMARD use,known serologies, presence of SE alleles, and C-reactive protein (CRP)levels in reduced and fully adjusted models, anti-PAD2 antibodies wereindependently associated with fewer swollen joints on baseline 28- and44-count joint exams (FIG. 2A and B). Similarly, after adjusting forage, smoking history, known RA serologies, DAS28, and current biologicuse, anti-PAD2 antibodies remained strongly and significantly associatedwith a lower frequency of ILD (_(adj)OR=0.24; p=0.017) (FIG. 2D). Thesedata suggest that the effect of anti-PAD2 on joint inflammation and ILDis not due to treatment with biologic DMARDs or other confoundingclinical variables.

TABLE 1 Characteristics of RA patients according to anti-PAD2 antibodystatus Anti-PAD2 Anti-PAD2 Negative Positive p- n = 150 n = 34 valueDemographic features Age, years, mean ± SD 61 ± 8 63 ± 9 0.28 Malegender, n (%) 68 (45)  6 (18)  0.003 Caucasian, n (%) 131 (87)  28 (82)0.44 Ever smoking, n (%) 88 (59) 21 (62) 0.74 Current smoking, n (%) 18(12) 2 (6) 0.38 Serologic and genetic features RF positivity > 40 units,n (%) 96 (64) 22 (65) 0.94 Anti-CCP positivity > 113 (76)  26 (76) 0.9420 units, n (%) Anti-PAD3/4 XR positivity, 17 (11) 3 (9) 0.66 n (%) AnyHLA-DRB1 SE alleles, 110 (74)  18 (53) 0.014 n (%) Clinical features RAduration, years, median 8 (4-17)  9.5  0.089 (IQR) (7-19) DAS28, median(IQR) 3.3 (2.5-4.0) 3.2 (2.5-3.9) 0.47 HAQ score (0-3), median 0.75(0.12- 1.0 (0.12- 0.19 (IQR) 1.44) 1.50) CRP, mg/L, median (IQR) 2.9(1.0-7.2) 3.3 (1.0-7.3) 0.91 Swollen joint count, median 4 (2-8)   2(1-6)    0.049 (IQR) Tender joint count, median 5 (2-14)  5 (2-12)  0.88(IQR) Nodules, n (%) 30 (21) 3 (9) 0.13 Any ILD, n (%) 50 (36)  5 (18) 0.060 Total SvdH Score, median 7 (1-42)  12 (0-55)   0.91 (IQR) Totalerosion score, median 3 (0-14)  3 (0-21)  0.83 (IQR) Total JSN score,median (IQR) 5 (0-27)  8 (0-28)  0.69 Δ SvdH Score (per year), 0.34  0(0-1.18) 0.15 median (IQR)* (0-2.1) Any increase in SvdH 69 (58) 13 (43)0.15 score, n (%)* Current treatment Non-biologic DMARDs, n (%) 123(83)  31 (91) 0.30 Biologic DMARDs, n (%) 64 (43) 19 (56) 0.17Glucocorticoids, n (%) 60 (40) 13 (38) 0.85 Cumulative prednisone, 3.2(0.5-8.7) 3.0 (0-11.7)  0.63 g, median (IQR) SD = standard deviation;IQR = interquartile range; RF = rheumatoid factor; DAS = diseaseactivity score; HAQ = health assessment questionnaire; CRP = C-reactiveprotein; SvdH = Sharp van der Heijde *Follow-up radiographs wereavailable in n = 149

TABLE 2 Associations of anti-PAD2 antibody levels with patientcharacteristics Median (IQR) anti-PAD2 Level in Patient Spearman'sWithout Characteristic With Characteristic Rho p-v 

  Demographic features Age, years 0.183 0.0 

  Male gender 1.94 (0.99-4.54) 1.36 (0.80-2.78) 0.0 

  Caucasian 2.39 (1.53-3.65) 1.58 (0.84-3.35) 0.0 

  Ever smoking 1.97 (0.99-3.26) 1.47 (0.85-3.39) 0. 

   Current smoking 1.85 (0.95-3.41) 0.90 (0.62-3.05) 0.0 

  Serologic and genetic features RF positivity 1.57 (0.74-3.40) 1.80(0.95-3.35) 0. 

   Anti-CCP positivity 1.39 (0.79-3.20) 1.86 (0.92-3.40) 0. 

   Anti-PAD3/4 XR positivity 1.64 (0.87-3.42) 1.65 (0.93-2.38) 0. 

   Any HLA-DRB1 SE alleles 2.41 (1.06-5.46) 1.54 (0.86-3.11 0.0 

  Clinical features RA duration, years 0.135 0.0 

  DAS28 −0.003 0. 

   HAQ score (0-3) 0.097 0. 

   CRP, mg/L 0.029 0.7 

  Swollen joint count −0.106 0. 

   Tender joint count −0.026 0. 

   Nodules 1.75 (0.93-3.40) 1.86 (0.69-2.67) 0. 

   Any ILD 1.85 (0.98-3.42) 1.46 (0.71-2.73) 0.0 

  Total SvdH Score 0.022 0. 

   Total erosion score −0.012 0. 

   Total JSN score 0.055 0. 

   Δ SvdH Score (per year) −0.027 0. 

   Any increase in SvdH score 1.88 (0.71-4.78) 1.78 (0.95-3.21) 0. 

   Current treatment Non-biologic DMARDs 1.58 (1.06-2.59) 1.71(0.87-3.35) 0. 

   Biologic DMARDs 1.54 (0.77-3.06) 1.92 (1.20-3.96) 0.0 

  Glucocorticoids 1.68 (0.92-3.65) 1.64 (0.84-3.21) 0. 

   Cumulative prednisone, g −0.020 0. 

   IQR = interquartile range; RF = rheumatoid factor; DAS = diseaseactivity score; HAQ = health assessment questionn 

  CRP = C-reactive protein; SvdH = Sharp van der Heijde

indicates data missing or illegible when filed

Anti-PAD2 Antibodies are Inversely Associated with the Progression ofJoint Disease

Since anti-PAD2 antibodies were independently associated with fewerswollen joints at baseline, they may also be independent markers of aless severe or less progressive arthritis phenotype. This hypothesis issupported by the finding that the average SJC, measured at threedistinct time points throughout the duration of the study, wassignificantly lower in patients with anti-PAD2 antibodies, even inreduced and adjusted multivariable models (FIG. 3A). Furthermore, theyearly change in SHS, a radiographic measure of joint damage, wasnegatively associated with anti-PAD2 antibody level where, on average,each anti-PAD2 unit was associated with 0.08 SHS unit per year lowerrate of radiographic progression (i.e. β=−0.08; p=0.028) (FIG. 3B). In amultivariable model adjusting for average CRP level, baseline SHS, andbaseline adiponectin level, each log unit higher level of anti-PAD2 wasassociated with a 9% lower odds of radiographic joint diseaseprogression (adjOR=0.91; p=0.016) (FIG. 3C). Importantly, biologicDMARDs were not associated with protection from progression ofradiographic joint damage in the univariate models, suggesting that theassociation of anti-PAD2 antibodies with less progressive joint diseaseis independent of treatment with biological DMARDs.

The Effect of Anti-PAD2 Antibodies on PAD2 Enzymatic Activity in Vitro

To define the potential mechanism whereby anti-PAD2 antibodies maycontribute to a milder RA phenotype, two assays were established todirectly address whether the antibodies inhibit PAD2 enzymatic activity.First, the effect of anti-PAD2 antibodies on PAD2-mediatedcitrullination of fibrinogen, a well-established extracellular substrateof PAD2 (Damgaard et al., Arthritis Res Ther, 16(6):498 (2014)), wasdetermined. Second, the ability of anti-PAD2 antibodies to modulatePAD2-mediated citrullination of cellular substrates in HEK293 celllysates was explored, as this may mimic PAD2-induced extracellularcitrullination of proteins released from dying cells. Interestingly, IgGpurified from patients with anti-PAD2 antibodies did not alter PAD2enzyme activity relative to IgG from anti-PAD2 negative individuals whenassessed for citrullination of purified fibrinogen (FIG. 4A) or HEK293cell lysate (FIG. 4B).

Discussion

Prognostic biomarkers for assigning risk of specific clinical outcomesin patients with RA are not currently available for use in clinicalpractice. Anti-PAD2 antibodies may represent such a clinical biomarkeras they are not associated with traditional genetic or serologic RA riskfactors, including SE alleles, ACPAs, and RF. Instead, patients withantibodies to PAD2 had fewer swollen joints on exam, less evidence ofradiographic ILD, and experienced less progression of radiographic jointdamage. The ability to identify patients who may have less severe lungand joint disease is an important step toward the management of patientswith RA, including minimizing risk of exposure to therapeutics withpotentially dangerous side effects and lowering overall health carecosts.

From a mechanistic perspective, the negative association of anti-PAD2antibodies with SE and lack of association with previously reportedserologies, suggests a unique mechanism for the development of theseantibodies. Future studies are needed to determine if there are specificgenetic, stochastic, or environmental factors that lead to the developedof anti-PAD2 antibodies in patients with RA. Importantly, PAD2 is highlyexpressed in neutrophils (9), the most abundant inflammatory cell typein the RA joint. Malinin et al., Am J Clin Pathol, 47(2):203-208 (1967).Factors that contribute to neutrophil damage or hyperactivation maypromote an abnormal release of PAD2 into the extracellular environment,which could lead to the development of anti-PAD2 antibodies. In thisregard, PAD2 is found extracellularly in RA synovial fluid likelyreleased from damaged or dying cells (Kinloch et al., Arthritis Rheum58(8):2287-2295 (2008)), and a recent report demonstrated that PAD2 isspontaneously secreted by neutrophils into the extracellular space. Zhouet al., Front Immunol, 8:1200 (2017). These mechanisms may thereforecontribute to increasing PAD2 antigen load in the RA synovialmicroenvironment and promote the generation of anti-PAD2 autoantibodies.

The findings that anti-PAD2 antibodies are associated with lessinflammatory and progressive joint disease and a lower frequency ofCT-ILD sets them apart from other RA autoantibodies described to date.Interestingly, unlike the previously described agonistic PADS/4 crossreactive antibodies, we found that anti-PAD2 IgG had no effect on thecatalytic activity of PAD2. While we cannot exclude the possibility thatthese findings result from limitations in our in vitro assays, it ispossible that anti-PAD2 antibodies exert protective effects in RA viamechanisms besides direct inactivation of the enzyme. For example, theseantibodies may promote more efficient clearance of extracellular PAD2 byphagocytes, which may decrease the enzyme concentration in therheumatoid joint, thereby reducing pathogenic PAD2-mediatedcitrullination.

The observational nature of the ESCAPE RA cohort and long averagedisease duration preclude us from answering questions related totreatment response outcomes and the prognostic potential of anti-PAD2antibodies in early RA. Further studies with longitudinal assessment ofPAD2 autoantibody levels and clinical trials assessing the role ofanti-PAD2 antibodies in predicting treatment response to specific DMARDsare warranted. The discovery of anti-PAD2 antibodies that are notassociated with traditional RA risk factors but are associated withfewer swollen joints, less radiographic ILD, and less progressive jointdamage has important prognostic and mechanistic implications in RA.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material cited herein areincorporated by reference. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood there from. The inventionis not limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

What is claimed is:
 1. A method for obtaining a prognosis for a subjecthaving rheumatoid arthritis (RA) comprising: a) obtaining a biologicalsample from a subject having RA; b) providing a substrate having a firstcapture probe bound thereto, wherein the capture probe comprisespeptidyl arginine deiminase 2 (PAD2) protein or a portion or fragmentthereof which comprises an antigen recognized by autoantibodies presentin subjects suffering from RA; c) contacting the substrate having thecapture probe bound thereto with the biological sample from the subject;d) measuring the amount of a complex of the capture probe and theautoantibodies formed by step c); e) providing a reference level sample;f) comparing the amount of a complex of the capture probe and theautoantibodies formed from the subject having RA to the amount of acomplex of the capture probe and the autoantibodies formed from thereference level sample; and g) identifying the subject as having a lowerrisk of developing a severe form of RA when the amount of a complex ofthe capture probe and the autoantibodies formed from the subject havingRA is increased compared to the amount of a complex of the capture probeand the autoantibodies formed from the reference level sample.
 2. Themethod of claim 1, wherein the method is an enzyme immunoassay method.3. The method of claim 1, wherein the method is a radioimmunoassaymethod.
 4. The method of claim 1, wherein the method is animmunoblotting method.
 5. The method of claim 1, wherein the method is achemiluminescent immunoassay (CIA) method.
 6. The method of claim 1,wherein the method is a particle based multi-analyte test (PMAT).
 7. Themethod of claim 1, wherein the capture probe is bound to the substratewith a binding reagent.
 8. The method of claim 1, wherein the biologicalsample is a blood, plasma, or serum sample.
 9. The method of claim 1,wherein the subject is human
 10. The method of claim 1, furthercomprising providing a less aggressive form of treatment of RA to thesubject identified as having a lower risk of developing a severe form ofRA.
 11. The method of claim 1, wherein the subject identified as havinga lower risk of developing a severe form of RA has a decreased risk ofdeveloping interstitial lung disease.
 12. A method of treating RA in asubject, comprising administering a therapeutically effective amount ofan antibody or fragment thereof that specifically binds to peptidylarginine deiminase 2 (PAD2) protein to the subject.
 13. The method ofclaim 12, wherein the antibody is a monoclonal antibody.
 14. The methodof claim 12, wherein the subject is human.
 15. The method of claim 14,wherein the antibody is a humanized antibody.
 16. The method of claim12, wherein the antibody is administered together with apharmaceutically acceptable carrier.
 17. The method of claim 12, whereinthe RA is severe RA.
 18. A kit comprising a substrate having a captureprobe comprising PAD2 protein or a portion or fragment thereof boundthereto, and a package for holding the capture probe and othercomponents for obtaining a prognosis for a subject having RA using themethod of claim
 1. 19. The kit of claim 18, further comprisinginstructions for using the kit to obtain a prognosis for a subjecthaving RA.
 20. The kit of claim 19, where the components andinstructions are for an enzyme immunoassay method, a chemiluminescenceassay (CIA) method, a fluorescent immunoassay, or a particle basedmultianalyte test (PMAT).
 21. The kit of claim 18, wherein the captureprobe is bound to the substrate with a binding reagent.